Enhanced T cell responsiveness to citrulline-containing ... - Asamnet

Multiple Sclerosis (2000) 6, 220±225
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Enhanced T cell responsiveness to citrulline-containing myelin basic protein
in multiple sclerosis patients
Laura R Tranquill 1 , Ligong Cao 2,3 , Nicholas C Ling 5 , Hubert Kalbacher 6 , Roland M Martin 1 and
John N Whitaker* ,2,3,4
1 Neuroimmunology Branch, NINDS, NIH, Bethesda, Maryland, MD 20892-1400 USA; 2 Department of Neurology,
University of Alabama at Birmingham, Birmingham, Alabama, AL 35233-7340 USA; 3 Center for Neuroimmunology of
the University of Alabama at Birmingham, Birmingham, Alabama, AL 35294 USA; 4 Neurology and Research Services of
the Birmingham Veterans Medical Center, Birmingham, Alabama, AL 35233 USA; 5 Neurocrine Biosciences, San Diego,
California, CA 92121 USA; 6 Physiologisch-Chemisches Institut der UniversitaÈt, TuÈ bingen University Medical School,
72076 TuÈ bingen, Germany
Myelin basic protein (MBP), a candidate autoantigen in multiple sclerosis (MS), exists in different isoforms and charge isomers generated by
differential splicing of exons and by a combination of posttranslational modi®cations, respectively. These various isoforms and charge isomers of
MBP vary in abundance and most likely serve different functions during myelinogenesis and remyelination. The least cationic among the charge
isomers of MBP is citrullinated and is referred to as MBP-C8. MBP-C8 is relatively increased in the population of MBP isomers in more
developmentally immature myelin and in MS brain tissue. In a previous study, we found that MBP-C8-reactive T cells could be detected in CD4+ T
cell lines (TCL) generated with MBP from both MS patients and normal controls. Here, we examined the frequency and peptide speci®city of MBP-
C8-speci®c TCL generated with MBP-C8 in MS patients and controls. Ten subjects grouped in ®ve sets, each an MS patient and a control, were
studied. In all cases, the MS patient had either a higher overall number of MBP-C8-responding lines, responded with greater sensitivity to the MBP-
C8 antigen or both. Few lines responded to the MBP-C8 peptides but, if they did, they appeared to be speci®c to the carboxyl-half of the MBP-C8
molecule. Given the large amounts of citrullinated MBP in MS brain tissue, a preferential T cell response to MBP-C8 may be involved in the
induction and perpetuation of this disease.
Multiple Sclerosis (2000) 6, 220±225
Keywords: multiple sclerosis; MBP; citrulline; T cells; human; isomer
Introduction
MS, the most frequent in¯ammatory demyelinating
CNS disease in humans, is considered a T cellmediated
autoimmune disease. 1 Its pathogenesis is
most likely multifactorial and involves both genetic
predisposition and precipitating environmental factors.
MBP, which constitutes approximately 30% of the
total CNS myelin protein, has been studied extensively
as a possible autoantigen in MS. 1 There are four major
isoforms of human MBP. The 18.5 kDa isoform is the
most abundant in humans. 2 In addition to size
heterogeneity, the 18.5 kDa isoform of MBP has at
least six charge isomers which can be resolved by
cation- or anion exchange chromatography at alkaline
pH 3 or according to net positive charge respectively. 4,5
These charge isomers are designated C1, the most
cationic, C2 ± 5 which are progressively less cationic
by one charge, and C8, the least cationic and the most
modi®ed. 5 Posttranslational modi®cations of the
charge isomers include phosphorylation, deamidation,
C-terminal arginine loss, and the presence of citrulline
*Correspondence: JN Whitaker, Department of Neurology,
University of Alabama at Birmingham, 619 19th Street South,
JT 1205, Birmingham, Alabama, AL 35249-7340, USA
Received 12 January 2000; accepted 4 April 2000
substituted for arginine at de®ned sites in the NH 2- and
COOH-terminal portions of the molecule. 5
In the charge isomer, MBP-C8, six arginines are
replaced by citrulline resulting in the loss of six
positive charges. In the human MBP molecule of 170
amino acid residues, the arginine to citrulline change
occurs on residues 25, 31, 122, 130, 159 and 170. 4 The
removal of positive charges alters the interaction of
MBP with membrane-embedded acidic lipid moieties. 6
Stoner 7 proposed a model that suggested that certain
arginine residues of the MBP molecule stabilize its
loop structure by ionic interactions with carboxyl and
phosphoryl groups, and that by substitution of
citrullines in MBP-C8, a destabilization of the loop
structure and possibly a conformational change in the
MBP molecule may result. In a study of the formation
of myelin crystalline multilayers, Moscarello 8 observed
that the denser and less compact myelin showed an
increased amount of MBP-C8 and a relative de®ciency
of MBP-C1. These authors suggest that the more MBP-
C1 in myelin, the more compact the myelin layers.
Moscarello 9 has also determined that the myelin
obtained from MS patients appears to be in a
developmentally less mature stage with the percentage
of MBP-C8 increased in MS white matter. This MBP-
C8-rich, developmentally immature myelin may be

more susceptible to degradation since it is less
compact than mature myelin and cannot form as
highly a structured myelin sheath as MBP-C1. The
resulting myelin instability may promote myelin
breakdown and thus either initiate and/or perpetuate
autoimmune T cell responses against CNS myelin. 9 In
fact, citrullination of MBP increases the speed of its
degradation by cathepsin D. 10 Alternatively, as the
number of attacks increase, relatively more MBP-C8
may be formed; MBP-C8 becomes available to the
immune system and its increased incidence may
perpetuate disease.
The role of MBP is still controversial in MS since
most of the evidence in humans is indirect. CD4+
MBP-speci®c T cells are often cytotoxic, 11,12 directed
against immunodominant regions of MBP which are
also encephalitogenic in rodent strains, 13 ± 15 often
express a Th1-type phenotype, 16 and are frequently
restricted by MS-associated HLA-DR molecules. 13,14
Currently, the role of MBP or also other myelin or
glial components such as proteolipid protein (PLP),
myelin oligodendrocyte glycoprotein (MOG), or aB
crystalline, to name only a few, are being evaluated. 1
In addition, other size isoforms and charge isomers of
MBP have also been examined both in EAE 17,18 and in
MS patients. 19,20 Due to the above reasoning, we have
been interested in MBP-C8 as a potential encephalitogen
17 and target antigen in MS. 21 Our data have
documented so far that MBP-C8 is indeed able to
induce EAE, 17 and a T cell response to MBP-C8 is
readily demonstrable in MS patients. 19 The current
study extends these previous observations by focusing
on the frequencies of MBP-C8-reactive T cells in MS
versus controls, and also initiates the attempts to
characterize the speci®city of the C8-reactive lines
using a panel of citrullinated MBP peptides.
Material and methods
Patients
Five male patients with de®nite MS, four with primary
progressive (PP), one with relapsing-remitting (RR)
MS, were investigated at the Neuroimmunology
Branch, NINDS, National Institutes of Health (NIH).
We attempted to examine patients with progressive
Table 1 Summary of clinical features and HLA haplotypes of MS patients
Sex
Age
MS type
Disease
Suration
Disability e
HLA-DR
disease since an accumulation of MBP-C8 in MS
brains with increasing disease duration had been
reported. 9 The research project was reviewed and
approved by the Institute Clinical Research Subpanel,
and informed consent was obtained from each patient.
MS patient demographics and clinical data are
summarized in Table 1. No patient was or had been
on immunomodulatory or immune suppressive treatment
at any time or had received glucocorticoids
within 90 days.
Cells
Peripheral blood lymphocytes (PBL) were isolated
from leukapheresis samples by Lymphocyte Separation
Medium (Organon Teknika, Durham, NC, USA).
After isolation, cells were cryopreserved in RPMI 1640
(Whittaker Bioproducts, Walkersville, MD, USA)
supplemented with 20% fetal calf serum (GIBCO,
Grand Island, NY, USA) or 20% human serum, and
10% dimethylsulfoxide (Sigma, St. Louis, MO, USA)
and stored in liquid nitrogen until use. HLA typing for
HLA-A, -B, -Cw, -DR, and -DQ was performed kindly
by T Simonis, Department of Transfusion Medicine,
NIH, Bethesda, MD, USA. HLA-DR and -DQ types are
summarized in Table 1.
Antigens
MBP-C1 was prepared from postmortem human
brain. 22,23 MBP-C8 was prepared from the pH3 extract
of human brain by a combination of cation-exchange
chromotography and high-pressure liquid chromatography
(HPLC) as described. 4 Citrulline-containing
MBP-peptides were made for regions containing citrulline
modi®cations. 19 Using a numbering system based
on 170 residues in MBP, the following three citrullinecontaining
peptides were synthesized as described: 24
18 ± 38 (ASTMDHACitHGFLPCitHRDTGIL) with citrulline
substitutions at positions 25 and 31, 115 ± 131
(SWGAEGQCitPGFGYGGCitA) with citrulline substitutions
at positions 122 and 130, and 151 ± 170
(SKIFKLGGCitDSRSGSPMARR) with a citrulline substitution
at position 159. No posttranslational modi®cations
other than citrullination were included in
the peptide sequences. All peptides were puri®ed by
using HPLC to a purity 495%.
MS1 MS2 MS3 MS4 MS5
M
43
PP a
SC c :1980
DX d :1988
8.0
B1*0101
B1*0405
M
59
PP
SX:1965
DX:1974
8.5
B1*0402
B1*1502
T cell response to MBP-C8 in MS
LR Tranquill et al
M
49
RR b
SX:1981
DX:1988
3.0
2(15),4
M
67
PP
SX:1959
DX:1965
7.0
2
M
49
PP
SX:1979
DX:1984
7.5
2(15),4
a b c d e
PP=primary progressive. RR=relapsing remitting. SX=onset of symptoms. DX=year of diagnosis. Disability expressed on
Expanded Disability Status Scale (Kurtzke, 1983)
221
Multiple Sclerosis

222
Multiple Sclerosis
Generation of MBP-C8-speci®c T cell lines (TCL)
PBL were seeded in complete media (Iscove's modi®ed
Dulbecco's medium with 2 mM L-glutamine, 50 mg/ml
gentamicin, 100 U/ml penicillin, and 100 mg/ml streptomycin-Whittaker
Bioproducts, Gaithersburg, MD,
USA) at 2610 5 cells/well into 96-well, U-bottom
microtiter plates (Nunc, Denmark) at 200 ml per well
and stimulated with 5 mg/ml of MBP-C8. Seven to 10
days later, IL-2 (Lymphocult-T HP, Biotest, Germany;
®nal concentration, 10 U/ml) was added. After another
7 to 10 days, cells were washed once within the plate
to remove lymphokines and resuspended in 200 ml of
complete medium. Fifty ml of the cell suspension were
transferred into three adjacent wells of a separate 96well,
U-bottom microtiter plate, and 150 ml of complete
medium containing 1610 5 autologous, irradiated
(3000 rad) PBL added. One well was stimulated with
5 mg/ml MBP-C8 and the other with 5 mg/ml MBP-C1.
Seventy-two hours later, 1 mCi of [ 3 H]thymidine
(Amersham, Arlington Heights, IL, USA) was added
for 4 h before [ 3 H]thymidine incorporation was measured
in a scintillation counter (Betaplate, Pharmacia,
Gaithersburg, MD, USA). The remaining wells in the
original plates were restimulated with 1610 5 autologous,
irradiated PBL and MBP-C8 at 5 mg/ml at the
same time the proliferation was analyzed. Wells with a
stimulation index (SI=c.p.m. of wells with antigen/
c.p.m. of wells without antigen) 42 and 41000 c.p.m.
in the proliferative assay were transferred into 24-well
plates (Costar, Cambridge, MA, USA), restimulated as
described above, and characterized for speci®city by
proliferative assay.
Proliferative assays
Proliferative assays were performed as described. 11
Brie¯y, 2610 4 line cells were incubated for 72 h with
5±10610 4 irradiated (3000 rad), autologous PBL as
antigen-presenting cells (APC) and two concentrations
(10 and 20 mg/ml ®nal concentration) of antigens or
peptides. One mCi of [ 3 H]thymidine (Amersham) was
added for another 4 h. Thymidine incorporation was
measured in a scintillation counter (Betaplate, Pharmacia,
Gaithersburg, MD, USA). Responses were
scored positive at an SI42 and 41000 c.p.m.
Results
CD4+ TCL were generated with MBP-C8 by seeding in
parallel PBL from ®ve MS patients and ®ve normal
controls in a standard split well limiting dilution
assay. During the split-well screening, which is
routinely performed between 14 and 17 days after
seeding, colonies are tested against MBP-C1, MBP-C8
and no antigen. Responses were scored positive at an
SI42 and 41000 c.p.m. The TCL were grouped in
three categories based on their proliferative responses
to the antigens: MBP-C8-responsive only, MBP-C1responsive
only as well as MBP-C8/MBP-C1-responsive.
As shown in Table 2, patient/control set number
1 showed the most impressive differences in the C8
Table 2 Summary of frequency and speci®city of MBP-C8 responsive T cell lines from MS patients and normals
Antigen
MBP-C1/MBP-C8
C1 C8 MBP-C8 only MBP-C1 only
Sets Patients Normal SI M # M # M # M #
1 MS1
2 MS2
3 MS3
4 MS4
5 MS5
ND1
ND2
ND3
ND4
ND5
T cell response to MBP-C8 in MS
LR Tranquill et al
53
55
53
55
53
55
53
55
53
55
53
55
53
55
53
55
53
55
53
55
6.0
7.4
4.3
0
17.4
29.8
13.5
16.7
6.1
11.4
4.6
7.8
7.1
7.1
0
0
193.3
193.3
6.2
8.5
12
7
1
0
2
1
7
5
10
3
4
1
3
3
0
0
2
2
8
5
7.9
8.9
4.4
0
20.7
20.7
12.5
26.4
5.1
6.9
4.1
5.0
11.7
11.7
0
0
81.5
120.7
6.4
7.8
20
16
1
0
2
2
9
5
9
4
3
1
4.7
8.2
0
0
16.1
22.3
5.8
5.8
SI: stimulation index; M: mean value of the stimulation indices; #: number of positive lines generated
3
3
0
0
3
2
8
5
6.2
7.7
0
0
0
0
0
0
5.4
7.2
9.6
9.6
7
2
0
0
6
4
3
3
3
2
0
0
0
0
0
0
2
1
1
1
4.8
0
0
0
3.1
0
4.0
0
4.9
6.2
0
0
0
0
3.1
0
0
0
0
0
1
0
0
0
1
0
1
0
5
3
0
0
0
0
3
0
0
0
0
0

esponse between the MS patient and the normal
donor. Sets 1 through 4 demonstrated a higher
percentage of MBP-C8-reactive lines in the MS patients
compared to the normal controls. In one normal donor,
number 5 (ND5), one more MBP-C8 only-reactive line
was established compared to the patient MS5. However,
the stimulation indices for the patient's MBP-C8
lines (both the MBP-C8/MBP-C1 and the MBP-C8 only
lines) had a mean value of 32.8 while the mean value
for the normal control was only 4.8. Thus, although
one more TCL had been generated from donor ND5,
their reactivities were not as strong as those from the
MS patient. Sets 1, 3 and 4 demonstrated a higher
percentage of MBP-C8/MBP-C1-reactive lines in the
MS patients than in the normal controls. However, in
sets 2 and 4, the lines that responded to both MBP-C1
and MBP-C8/MBP-C1 were more numerous in the
controls than in the patients.
Statistical analysis (t-test) was performed between
the TCL derived from MS patients and controls at each
cut-off value (SI greater than or equal to 3 and 5) for
the total number and the mean SI value of the three
groups of TCL: TCL responding to MBP-C8/MBP-C1, to
MBP-C8 only, or to MBP-C1 only. Further analysis was
performed to analyze the differences in the total
number of MBP-C8-reactive lines between MS patients
and controls. In each group, the mean number of MBP-
C8-responding lines in the MS patients was higher (in
many cases as much as three times higher) than the
mean for the controls both with respect to mean SI and
number of responding lines. However, due to the
limited number of data sets, our sample size was too
small to reach statistical signi®cance, but remained a
trend.
In order to test whether the trend towards a stronger
MBP-C8-speci®c response was also re¯ected in the
magnitude of the proliferative response of individual
lines, we analyzed the data from these 10 individuals
Figure 1 Con®rmatory testing for antigen-speci®c response
of MBP-C8-elicited T cell lines. The MBP-C8-responsive T
cell lines were generated as described in Materials and
methods section. The proliferative responses to MBP-C8 of
these T cell lines at short term (after three cycles of antigen
stimulation) and long term (after more than ®ve cycles of
antigen stimulation) were compared
T cell response to MBP-C8 in MS
LR Tranquill et al
with respect to the stimulation indices for the positive
lines. The number of positive lines for each individual
was determined using more stringent criteria, i.e. cutoff
values for the SI of greater or equal to 3 and then
again with a value of greater or equal to 5. In sets 1, 2,
3 and 5, the numbers of MBP-C8 only-reactive lines
were higher in the MS patients than in controls at both
cut-off levels. Set 4 (both patient and control) failed to
show any MBP-C8 only-reactive lines at the 43 or45
SI cut-off values. Using these criteria for the MBP-C8/
MBP-C1 lines, sets 1, 3 and 4 demonstrated a higher
number of positive lines at both SI values in the
patients as compared to the controls. In contrast, sets 2
and 5 had higher numbers of positive lines at both SI
cut-off values in the normal individuals as compared
to the MS patients. There is not only a trend towards
higher numbers of MBP-C8-responding lines in MS
patients versus controls, but these also exhibited
higher SIs than those of the control individuals.
A large proportion of the highly MBP-C8-responsive
lines remained antigen-speci®c upon con®rmatory
testing (Figure 1) using the original MBP-C8 antigen.
In an attempt to map the epitope speci®city of MBP-
C8-speci®c TCL to individual citrulline residues, the
MBP-C8-positive lines were tested against a panel of
three MBP-C8 peptides (Figure 2) corresponding to the
three regions of MBP where the citrulline modi®cations
occur. Of the 11 MS1 MBP-C8-positive lines
tested against whole MBP-C8, MBP-C1, and the three
MBP-C8 peptides, all 11 recognized whole MBP-C8, 10
recognized MBP-C1 and three recognized MBP-C8
peptides. As shown in Figure 2, TCL 1C8 gave an SI
of 21.3 for whole MBP-C8, an SI of 2.1 for MBP-C1,
and an SI of 19.5 for MBP-C8 peptide 151 ± 170. MS1
TCL 3A5 responded with an SI of 114.3 to MBP-C8, an
SI of 29.1 for MBP-C1, and an SI of 75.2 for MBP-C8
peptide 151 ± 170. MS1 line 2A4 responded with an SI
of 103.9 to MBP-C8, 150.0 to MBP-C1, and 2.2 to MBP-
Figure 2 Proliferation assay for T cell response to MBP
isomers and MBP-C8 peptide. Eleven MBP-C8 generated T
cell lines from two MS patients were examined for their
responses to MBP-C8 peptides. The heterogeneity in epitope
speci®city among these T cell lines was evident and three
typical patterns of epitope recognition are shown in the
®gure
223
Multiple Sclerosis

224
Multiple Sclerosis
C8 peptide 115 ± 131. Further attempts to map the
peptide speci®city to individual citrulline residues
were not successful.
Discussion
T cell response to MBP-C8 in MS
LR Tranquill et al
Many potential candidate autoantigens, particularly
MBP, PLP and MOG, have been studied for their
relevance in MS and encephalitogenicity in EAE
models. 1 Assessing biological relevance in humans has
to rely on indirect evidence such as increases in T cell
frequency, higher af®nity for the antigen, memory
phenotype, cytokine pro®le, i.e. secretion of proin¯ammatory
cytokines, and cytotoxicity. The results of the
present study demonstrate that the T cell response
against a post-translational modi®cation of MBP, i.e.
MBP-C8, is relatively enhanced in MS patients as
compared with HLA-DR-matched control donors. Due
to the limited availability of MBP-C8, a larger cohort of
MS patients could not be studied, and it needs to be
stressed that our current results have been obtained with
MS patients with long-standing disease and an overrepresentation
of PP-MS. The increased response in MS
patients is evident in some cases as a higher total
number of MBP-C8-reactive TCL and, in other cases, as
higher stimulation indices in the reactive lines or both.
In addition, they were tested for reactivity against three
citrullinated peptides of MBP. TCLs derived from
patient MS1 showed a predominant reactivity to the Cterminal
MBP-C8 peptide 151 ± 170. We were unable to
map further the ®ne speci®city in the other two MBP-C8
peptides. Furthermore, our overall limited success with
determining the peptide speci®city may indicate either
that the MBP-C8 isolated from native brain myelin
contains other important post-translational modi®cations
which were not present in the synthetic, citrullinecontaining
peptides, or that the natural proteolytic
cleavage of MBP-C8 in antigen-presenting cells creates
different peptides than the ones chosen for synthesis.
The relatively higher level of MBP-C8 in MS brain 9
could help explain our ®nding of a prominent role for
MBP-C8 as an antigen for T cells in MS. The
continuous or repeated release of MBP-C8 and its de
novo synthesis could lead to an in vivo expansion of
MBP-C8-speci®c T cells. The trend for an overall
greater response to MBP-C8 in MS patients (with
respect to both number of MBP-C8 responsive TCL
and magnitude of their SIs) supports these considerations.
Besides an overall stronger response to MBP-C8 in
MS patients, it is also noteworthy that the MBP-C8speci®c
response exceeded that to MBP-C1 considerably,
e.g. in patient MS1. Whether this is due to a
higher abundance of MBP-C8 in this patient or higher
immunogenicity of MBP-C8 for T cells in this
individual is unresolved. The latter possibility may
be due, in part, to the fact that certain native MBP
isoforms are expressed in the thymus where tolerance
may be established. 25 It is not known if central, i.e.
thymic, tolerance to the developmentally more immature
MBP-C8, occurs.
Similar to other isoforms of MBP, e.g. the 21.5 kd
MBP in which exon 2 is expressed, MBP-C8 may be
relevant to disease pathogenesis not only since its
expression is upregulated during the disease process,
but also due to the physicochemical instability of
MBP-C8 compared to the more basic MBP charge
isomers. In our previous report, 10 under the condition
of enzymatic degradation, MBP-C8 is degraded much
faster by cathepsin D than MBP-C1 to release
immunodominant epitopes which presumably prime
not only MBP-C8-reactive T cells but also MBP-C1responsive
T cells. This was proved by the ®ndings of
the current experiment in which some human T cell
lines generated with MBP-C8 react with both MBP-C8
and MBP-C1 and to our surprise, some even respond
to MBP-C1 only. With respect to MBP-C1-only reactive
lines, it is most likely that the absence of reactivity to
MBP-C8 which had been used to generate the lines
initially, is due to differences in the relative sensitivity
towards the two antigens, i.e. MBP-C1 induces a
heteroclitic response, causing stimulation at lower
antigen doses compared to MBP-C8 as previously
reported. 26 In two of three multiplex families studied
there was a signi®cantly increased exon 2 MBPspeci®c
response even though there was no difference
in the frequency of exon 2 MBP-speci®c T-cell
between the MS group and the healthy control group. 20
The T cell response to the MBP-C8 isomer is
elevated in MS patients, but clearly also, though in a
smaller scale, present in healthy controls. Future
studies will need to address whether increased
expression and immunoreactivity to MBP-C8 correlates
with speci®c disease courses and subtypes of MS in
larger groups of patients strati®ed for these criteria. In
addition, cytokine pro®les of the MBP-C8 T cell lines
should be examined in the future to assess for
differences between the MS patients and the normal
controls. The encephalitogenicity of MBP-C8, its
structural instability and the results of the present
investigation underscore the prominent role for a
selective charge isomer of MBP. Other size isomers
and post-translational modi®cations of MBP may
deserve more detailed analysis for their roles in the
autoimmune response to MBP.
Acknowledgements
This investigation was supported by the Research
Program of the Veterans Administration. Ms Linda
Brent and Mrs Denise Ball assisted in the preparation
of the manuscript.
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